This project proposes a series of continuous wave and advanced EPR studies of mutant and PEGylated hemoglobins (Hbs) whose results will aid in the optimization of new design of blood substitutes. The first of these investigations addresses the intrinsic stability of the Hb moiety of these molecules towards autoxidation and subsequent denaturation to form hemichromes, both reversible or reconstitutable, and irreversible. Included will be in vitro studies of identification and kinetics of formation as well as investigations of Hb stability in animal models. A second series of experiments addresses specific aspects of the reaction of NO and hemoglobin, with reference to distance measurement of heme to non-heme binding sites and the role of protein in governing the nature of these sites. Low-temperature photolysis of Co(II)O2, Fe(II)NO, and Co(II)NO forms of Hb-based blood substitutes, followed by EPR, will be used to measure distances between photolyzed ligand and metal centers. Mixed-metal Fe-Co complexes will be examined to delineate differences in alpha and beta chains in accommodating NO away from heme. T1 measurements using spin echo-detected saturation recovery techniques are planned with Fe(II)NO Hb and NO, while ESEEM studies will be carried out with Fe(II)NO Hb and deoxyCo(II) Hb containing 13CO. A third set of studies planned of a series of singly and doubly spin-label PEGylated Hbs are to measure distances between the Hb and appended PEG moieties using the double electron-electron resonance (DEER) method. These experiments will be used to test a hypothesis concerning the effect of PEGylation on the intrinsic viscosity as due to the entrapment of a water channel between the appended PEG and the Hb moiety.